Renewable biomass, including lignocellulosic material and agricultural residues such as corn fiber, corn stover, corn cob, wheat straw, rice straw, and sugarcane bagasse, are low cost materials for bioethanol production. However, significant challenges exist for sustainable and efficient conversion of biomass to ethanol. One barrier for the conversion of biomass to ethanol is the stress conditions involved in the biomass pretreatment process. Dilute acid hydrolysis is commonly used in biomass degradation which hydrolyzes cellulose and hemicellulose fractions to increase fiber porosity to allow enzymatic saccharification and fermentation of the cellulose fraction. (Saha, B. C., et al., 2003. J. Ind. Microbiol. Biotechnol., 30:279-291). However, acid hydrolysis of biomass generates inhibitory compounds that interfere with microbial growth and hinders subsequent fermentation. For example, the resultant hydrolysate from dilute acid pretreatment comprises of a complex mixture, in which more than 35 potentially toxic ethanologenic inhibiting compounds have been identified. (Luo, C., et al., 2001. Biomass Bioenergy 22:125-138). These compounds can be divided into four main groups of aldehydes (such as furfural, 5-hydroxymethylfurfural, etc.), ketones, phenols, and organic acids (such as acetic, formic, levulinic acids, etc.). A remeditation process is needed to remove the inhibitors before the hydrolysate can be used for microbial growth and fermentation
Two of the most potent inhibitors are furfural and 5-hydroxymethylfurfrual (5-hydroxymethylfurfrual referred to as “HMF” hereafter). During sugar degradation, pentose dehydration leads furfural build up while hexose dehydration leads to HMF build up. These two inhibitory compounds reduce enzymatic biological activities, break down DNA, inhibit protein and RNA synthesis (Modig, T., et al. 2002. Biochem J., 363:769-776). Yeast can be repressed by the inhibitory complex as low as a 5 mM combination of furfural and HMF (Liu, Z. L., et al., 2004. J Ind Microbiol Biotechnol., 31:345-352). Most yeast strains, including industrial strains, are susceptible to the complexes associated with dilute acid hydrolysis pre-treatment (Martin, C., et al., 2003. Enzy. Micro. Technol., 32:386-395). Yet few yeast strains tolerant to inhibitors are available and the need for tolerant strains is well recognized (Klinke, H. B., et al., 2004. Appl. Microbiol. Biotechnol., 66:10-16 and Zaldivar J., et al., 2001. Appl. Microbiolo. Biotechnol., 697 56:17-34).
To facilitate fermentation processes using existing yeast strains, additional remediation treatments are required. Such treatments including physical, chemical, or biochemical detoxification procedures are utilized to remove these inhibitory compounds. For example, U.S. Pat. No. 4,461,648 describes a steam cooking method wherein lignocellulosic material is fed in a pressurized steam vessel and optimized with volatiles vented from the vessel. Additionally, U.S. Pat. No. 6,090,595 describes a pretreatment method of cellulosic feedstock wherein the ratio of [arabinan plus xylan] to [xylan plus arabinan plus cellulose] is utilized for ethanol production. Additional methods for hydrolysate detoxification include the addition of ion exchange resins (Nilvebrant, N. O., et al., 2001. Appl. Biochem. Biotechnol., 91/93:35 49), addition of active charcoal (Gong, C. S., et al., 1993. Appl. Biochem. Biotechnol., 39/40:83 88), enzymatic detoxification of hydrolysate using laccase and lignin peroxidase (Jonsson, L. J., et al. 1998. Appl. Microbiol. Biotechnol., 49:691-697), overliming (Martinez, A., et al. 2001. Biotechnol. Prog., 17:287-293), increasing yeast inoculum (Chung, I. S., et al. 1985. Biotechnol Bioeng., 27:308-315). However, these additional steps present additional complexity to the production of bioethanol production, produces waste products, and adds significantly to overall cost production. As such there is a need in to the art to engineer a yeast strain that is tolerant to inhibitors resulting from an economic hydrolysis pre-treatment process, circumventing remediation treatments.
It has been demonstrated that ethanologenic yeast individual strains of Saccharomyces cerevisiae can withstand and in situ detoxify furfural or HMF. (Liu, Z. L., et al., 2005. Appl. Biochem. Biotechnol., 121-124:451-460 and Liu, Z. L., et al. 2004. J. Ind. Microbiol. Biotechnol., 31:345-352). However, these strains are tolerant to a single inhibitor of either furfural or HMF, but not to both. As such, there is a need in the art for a Saccharomyces yeast strain that is tolerant of both inhibitors.